Team:Caltech/Protocols/rcsA Lysogen Induction

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Revision as of 10:17, 23 October 2008

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rcsA Lysogen Induction

Grow fresh overnight cultures of lysogens of bacteria carrying the rcsA construct.
Grow fresh overnight cultures wildtype E. coli.

Diagram of the induction process.

Make a 1:1000 dilution of lysogen culture and incubate the cultures until they reach an OD600 of 0.1
- More specifically, when the culture is swirled, cloudiness is observed. Check OD on plate reader as it is important to keep OD constant between trials and experiments.
At this time also make a 1:1000 dilution of the wildtype E. coli culture.
Add AHL to bring the concentration within the Lysogen culture to 10 nM.
Resume incubation at 37 degrees C for 1.5 hours.
At 1.5 hours, add 5% v/v formaldehyde to the lysogen culture and vortex vigorously.
Spin down cells at 5000xg for 5 minutes.
Remove an aliquot of the supernatant.
Follow the phage titering protocols using the supernatant as the phage solution, and titer against the culture of wildtype E. coli.
Count plaques the following day to estimate the concentration of phage in supernatant.

@@ Line 9: / Line 9: @@
 #Grow fresh overnight cultures of lysogens of bacteria carrying the rcsA construct.
 #Grow fresh overnight cultures wildtype E. coli.
-[[Image:Phage_titer.jpg|right|frame|An example plate. Dark spots are cleared zones in a lawn of bacteria.]]
+[[Image:Induction.png|right|thumb|400px|Diagram of the induction process.]]
 #Make a 1:1000 dilution of lysogen culture and incubate the cultures until they reach an OD600 of  0.1
 #* More specifically, when the culture is swirled, cloudiness is observed. Check OD on plate reader as it is important to keep OD constant between trials and experiments.